Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the United States. Because of its stubborn resistance to chemotherapy, there is an urgent need to develop novel therapeutic strategies for this deadly cancer. Numerous studies have revealed an important etiological contribution of activated mutations of K-ras and loss of function mutations of p16, p53 and Smad4. Mouse models that combine mutations in these genes have permitted their study specifically in the context of the pancreas thanks to the Cre/LoxP system. However, the potential role of other regulatory pathways, either with respect to the development of PDAC and/or in relation to the genesis of its marked chemoresistance, has not been thoroughly investigated. MicroRNAs (miRNAs) are a new class of short non-coding RNA genes [10-12], which act as post- transcriptional negative regulators of gene expression [13]. MiRNA-mediated regulation of tumorigenesis is emerging as a new paradigm in the field of cancer biology. The large number of predicted target genes for each miRNA suggests that miRNAs could provide a novel and global mechanism to modulate gene expression comparable to genome-wide epigenetic and transcriptional changes associated with cancer. Moreover, several reports indicate that specific miRNAs exhibit functions reminiscent of tumor suppressors and oncogenes. In addition, global decreases in miRNA activity by loss of Dicer or other enzymes required for miRNA processing result in enhanced tumorigenic potential, reflecting on the overall role of miRNAs in mediating tumor suppressive functions. Our preliminary studies and reports from other groups have linked a subset of miRNAs to PDAC based on: 1) their differential expression in pancreatic cell lines and/or in clinical specimens and/or 2) their interaction with signaling pathways etiologically relevant such as K-Ras, p16 and p53. Thus, miRNAs could serve as useful biomarkers for early detection, diagnosis and/or prognosis, advance our understanding of the molecular mechanisms of tumorigenesis, and serve as novel therapeutic targets. Based on these observations, our overall hypothesis is that miRNAs exert a crucial role in the initiation and progression of PDAC and that combinatorial therapy with current therapeutic agents and synthetic modulators of this novel class of regulatory RNAs may provide an effective strategy to increase patient response to treatment. We have established several transgenic murine strains that bear conditional mutations in K-ras and/or p16 genes, the most frequently mutated genes in PDAC, and we propose to conduct a functional analysis of miRNAs aimed at assessing: 1) The effect of decreased global miRNA activity in disease progression by crossing a conditional loss of function dicer1floxed/floxed animal to cancer-prone animals bearing mutated alleles of K-ras and/or p16 genes. 2) The contribution of individual miRNAs to pancreatic tumorigenesis in vitro by manipulating levels of miRNA expression/activity in murine and human pancreatic cancer cell lines. PUBLIC HEALTH RELEVANCE: Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy in which mortality virtually equals incidence, yet for which there are no satisfactory therapeutic treatments. Emerging evidence suggests a pivotal role of microRNAs in cancer biology. We hypothesize that combinatorial therapy of current chemotoxic compounds and synthetic modulators of this novel class of regulatory RNAs may provide an effective strategy to increase patient response to treatment. In this proposal, we will investigate the etiological contribution of microRNAs to PDAC and conduct a preliminary assessment of their potential therapeutic application.